Fig. 3: RapaBlock abolishes RapaLink-1-dependent weight loss, glucose intolerance, and liver toxicity. | Nature Communications

Fig. 3: RapaBlock abolishes RapaLink-1-dependent weight loss, glucose intolerance, and liver toxicity.

From: Brain-specific inhibition of mTORC1 eliminates side effects resulting from mTORC1 blockade in the periphery and reduces alcohol intake in mice

Fig. 3

a Timeline of experiment. Mice were treated three times a week with vehicle (white) RapaLink-1 alone (1 mg/kg, purple) or with a combination of RapaLink-1 (1 mg/kg, purple) and RapaBlock (40 mg/kg, pink) for 4 weeks, and body weight, glucose tolerance, and liver toxicity were evaluated. b Co-administration of RapaLink-1 and RapaBlock eliminates RapaLink-1-dependent body weight loss (Two-way ANOVA: effect of time (F11,312 = 3.467, p = 0.0001), the effect of treatment (F2,312 = 27.55, p < 0.0001) and interaction (F22,312 = 1.731, p = 0.0233); at day 8: vehicle vs. RapaLink-1 p = 0.0440, at day 9: vehicle vs RapaLink-1 p = 0.0061, at day 10: RapaLink-1 vs. RapaLink-1+RapaBlock p = 0.0249, at day 11: vehicle vs. RapaLink-1 p = 0.0003, RapaLink-1 vs. RapaLink-1+RapaBlock p = 0.0014, at day 12: vehicle vs. RapaLink-1 p < 0.0001, RapaLink-1 vs. RapaLink-1+RapaBlock p < 0.0001)). c Glucose tolerance test was performed during the last week of chronic drug treatment. Co-administration of RapaLink-1 and RapaBlock reduces RapaLink-1-dependent increase in blood glucose (two-way ANOVA: effect of time (F11,312 = 3.467, p = 0.0001), the effect of treatment (F2,312 = 27.55, p < 0.0001) and interaction (F22,312 = 1.731, p = 0.0233); at 20 min: vehicle vs. RapaLink-1 p < 0.0001, RapaLink-1 vs. RapaBlock p = 0.0008, at 30 min: vehicle vs. RapaLink-1 p < 0.0001, RapaLink-1 vs. RapaBlock p = 0.0008, at 60 min: vehicle vs. RapaLink-1 p = 0.0003, RapaLink-1 vs. RapaBlock p = 0.0250). d–h Co-administration of RapaLink-1 and RapaBlock protects against RapaLink-1-dependent liver toxicity. d–f The liver was dissected 24 h after the last drug administration and S6 and STAT3 phosphorylation were measured. d Representative images of pS6, total S6 (top panels), phospho-STAT3 (pSTAT), total STAT3 (middle panels), and actin (bottom panel). e Co-administration of RapaLink-1 and RapaBlock protects mTORC1 activity in the liver (One-way ANOVA: F2,15 = 42.77, P < 0.0001, r2 = 0.8508; vehicle vs. RapaLink-1 p < 0.0001, vehicle vs. RapaLink-1+RapaBlock p = 0.8715, RapaLink-1 vs. RapaBlock p < 0.0001). f RapaBlock reverses RapaLink-1-dependent increase in STAT3 phosphorylation (one-way ANOVA: F2,15 = 104.9, p < 0.0001, r2 = 0.9333; vehicle vs. RapaLink-1 p < 0.0001, vehicle vs. RapaLink-1+RapaBlock p = 0.6087, RapaLink-1 vs. RapaBlock p < 0.0001). g, h RapaBlock protects against RapaLink-1-dependent increase of fibrogenic markers, Timp1 (one-way ANOVA: F2,25 = 13.18, p = 0.0001, r2 = 0.3733; vehicle vs. RapaLink-1 p = 0.0004, vehicle vs. RapaLink-1+RapaBlock p = 0.5992, RapaLink-1 vs. RapaLink-1+RapaBlock p = 0.0041), and Col4a1 (one-way ANOVA: F2,25 = 7.446, p = 0.0029, r2 = 0.4605; vehicle vs. RapaLink-1 p = 0.0069, vehicle vs. RapaLink-1+RapaBlock p = 0.9960, RapaLink-1 vs. RapaLink-1+RapaBlock p = 0.0069). b, c Data are presented as mean ± SEM. Significance was determined using RM two-way ANOVA followed by Tukey’s multiple comparisons test. Vehicle, n = 9, RapaLink-1, n = 10; RapaLink-1+RapaBlock, n = 10, * = RapaLink-1 vs. Vehicle, # = RapaLink-1 vs. RapaLink-1+RapaBlock. * or # p < 0.05, ** or ## p < 0.01 and *** or ### p < 0.001, ns = non-significant. e, f Data are presented as the individual data points and mean densitometry values of the phosphorylated protein divided by the densitometry values of the total protein ± SEM and expressed as % of vehicle. Significance was determined using One-way ANOVA followed by Tukey’s multiple comparisons test. n = 6 per condition, ***p < 0.001, ns = non-significant. g, h Data are expressed as a ratio of TIMP1 or Col4a1 to total GAPDH and presented as individual data points and mean ± SEM. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test. Vehicle, n = 9, RapaLink-1, n = 9, RapaLink-1+RapaBlock, n = 10, **p < 0.01, ***p < 0.001, ns = non-significant.

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